Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation

Luke A. Gilbert, Max A. Horlbeck, Britt Adamson, Jacqueline E. Villalta, Yuwen Chen, Evan H. Whitehead, Carla Guimaraes, Barbara Panning, Hidde L. Ploegh, Michael C. Bassik, Lei S. Qi, Martin Kampmann, Jonathan S. Weissman

Research output: Contribution to journalArticlepeer-review

1019 Scopus citations

Abstract

While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%-99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.

Original languageEnglish (US)
Pages (from-to)647-661
Number of pages15
JournalCell
Volume159
Issue number3
DOIs
StatePublished - Oct 23 2014

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

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